Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) and generates phosphatidic acid (PA) and choline. PA has been implicated as a biologically active molecule and can be further metabolized by a specific hydrolase to form diacylglycerol (DAG), a protein kinase activator. Procedures for the purification of PLD from rat brains have been improved using extraction of brain membranes with 1% triton followed by chromatographies on heparin HPLC, Mono S and Mono Q columns. The partially purified rat brain membrane PLD can be activated in the presence of GTPgS by the members of ARF and Rho families as reported by others previously. Fractionation of rat brain cytosolic proteins on a Heparin-Sepharose and DEAE columns yielded multiple activity peaks when assayed for their capacity to activate the partially purified PLD in the presence of GTPgS. Several peaks were proved to contain ARF or Rho A proteins. ARF eluted as multiple peaks probably because it consists of multiple family members. In addition to the activity peaks attributable to ARF and Rho A, we identified another peak with distinct behavior. Further purification of this seemingly novel activator produced a protein of 21 kDa, which does not react with antibodies to ARF, Rho A or Rho B. Activation by this protein was as efficient as those by ARF and Rho family members and required the presence of PIP2. This suggests that brain PLD activity can be modulated by a variety of small molecular weight G (Smg) proteins.